Experiments were performed exactly as approved by the Animal Care and Use Committee at the University of Illinois College Of Medicine in Rockford, IL, and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Hi everyone. Epub 2012 Apr 11. Our study demonstrates a role of in vivo development of WT MEF-derived fat pads in preventing and rescuing the development and progression of long-term metabolic disease in the leptin-deficient ob/ob mouse model. It has been postulated that an ideal therapy would provide leptin endogenously (23–25). Although we cannot fully explain the variation, it could be caused by heterozygosity for the leptin allele. How to know that my primary osteoblast culture contaminated with fibroblast? MEF fat pads (0.79 ± 0.21 g) made up 2.2% of obR BW. is there any other way to check it? Data represented as the average per 12 hours and expressed as mean ± SEM (n = 4 to 5). 2006;38(7):1063-75. doi: 10.1016/j.biocel.2005.12.014.

(a) Oxygen consumption, (b) respiratory exchange ratio (RER), (c) energy expenditure, and (d) movement. © 2020 Springer Nature Switzerland AG. Importantly, before the onset of obesity, obR mice were protected from fat mass accumulation and hepatic steatosis. Lengner, C.J., et al. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S, Kern PA, Friedman JM.  |  Guide for the Care and Use of Laboratory Animals. Adipocyte size was measured and calculated in Adiposoft software (ImageJ) (33). Additionally, ob/ob mice have impaired glucose tolerance and insulin sensitivity, along with elevated levels of other hormones associated with insulin resistance, including insulin and gastric inhibitory polypeptide (GIP) (18–20). (b) Quantification of adipocyte area from H&E-stained sections of epididymal WAT and MEF fat pad. By Day 7, MEFs looked elongated and their pseudopodia grew across each other to form a confluent monolayer culture. Insulin has been shown to play an important role in the development of adipocytes, and it is possible that high insulin levels are necessary for the development of fat pads in vivo in the setting of obesity. Mouse Embryonic Fibroblasts (MEFs) are critical biological reagents in stem cell research. Cell viability at Day 1 and Day 7 was calculated as the ratio of total propidium iodide (PI) negative cells to total cells indicated by Hoechst nuclear staining. By contrast, hESC and mESC colonies grown on low-quality MEFs are less defined, less compact, and show indications of differentiation. After a total of 8 weeks after MEFs, obR mice had a 30% lower BW than ob/ob mice [Fig. 7(d)]. Search for other works by this author on: Department of Medical Education, College of Medicine, University of Central Florida, Orlando, Florida, Department of Medicine, Veterans Affairs St. Louis Healthcare System, John Cochran Division, St. Louis, Missouri, Prevalence of the metabolic syndrome in the United States, 2003–2012, American Heart Association Statistics Committee and Stroke Statistics Subcommittee, Heart disease and stroke statistics—2017 update: a report from the American Heart Association, Will all Americans become overweight or obese? First, we saw that obR mice had a dramatic 57% lower liver weight [Fig. Blackman B.E., et al. Remaining steps of the both procedures were the same. Significance determined by one-way ANOVA. performed initial experiments, interpreted data, assembled figures, and prepared the manuscript. Moon HS, Dalamaga M, Kim SY, Polyzos SA, Hamnvik OP, Magkos F, Paruthi J, Mantzoros CS. Preparation of primary mouse embryonic fibroblasts MEF cells are isolated from mouse embryos at day 14 of gestation as described by Robertson (1987): At animal facility: - Isolate embryos and wash once in PBS; dissect individual embryos to remove the head and soft tissues (liver, heart, and other viscera) and wash the carcasses twice in PBS. CVD is the leading cause of death in the United States, and the combined annual cost associated with diabetes and CVD is >$500 billion (2). De Matteis R, Dashtipour K, Ognibene A, Cinti S. Margetic S, Gazzola C, Pegg GG, Hill RA. Leptin-deficient humans and mice are obese, diabetic, and infertile and have hepatic steatosis. I found that people use mouse embryonic fibroblast from the KO mice to check or confirm the role of their proteins in the DDR (e.g. Furthermore, obRs have sustained metabolic protection and restoration of fertility. Nature 455:674–678, Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. It is also worth considering that ob/ob mice are hyperinsulinemic, providing another possible requirement for fat pad development. If anyone has had success using ear biopsies and 2-3 mm tail clips for primary cells, could you please share your experience and/or protocols? When examining WAT depots we found that obR mice had 86% lower inguinal (subcutaneous) [Fig. The typical MEF preparation time saved with Gibco MEFs. Food intake decreased in rescue mice. **P < 0.01; ****P < 0.0001. P < 0.05 was considered significant. Notably, WT MEF-derived fat pads were the result of a single injection, and fat pad mass was ~0.5 to 1 g in size, which was sufficient for ameliorating effects on BW and was sustained over time. Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. Furthermore, a single pregnant dam can provide embryos for MEFs to sufficiently rescue up to eight ob/ob mice. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error.

The simple and reliable procedure significantly improves the yield and overall cell health and is applicable to a variety of mouse strains, including pathogen-free mice and genetically modified disease mice.

7(c)]. Figure 5. As with our previous experiments, we found that obR liver weight was 46% less than that in ob/ob mice [Fig. Not logged in These cells are relatively simple to generate and are a powerful tool to explore regulatory networks, examine biochemical profiling of protein complexes, and investigate novel signaling pathways associated with innate immune system signaling. Birth Defects Res B Dev Reprod Toxicol. This site needs JavaScript to work properly. Originally described in 1949, ob/ob mice have a mutation in the leptin gene (Lep) resulting in the production of a nonfunctioning protein and are a valuable tool for the study of metabolic defects and therapies for leptin deficiency (5, 16). (c) Liver weight measured at necropsy. Mouse embryonic fibroblasts (MEFs) are stem cell-like fibroblasts that are widely used as feeder cells, since they secret various growth factors to support embryonic stem cells self-renewal and growth in an undifferentiated state.
I did primary culture of mouse choroid plexus epithelial cells. The equivalent of MEFs from one embryo was injected into each mouse with a 25-gauge needle. Not affiliated (a) Leptin levels in plasma of mice taken at terminal necropsy. Future studies may provide a better understanding of in vivo fat development and aid in the discovery of treatments for metabolic diseases. Next, we examined the effect of MEF transplantation on glucose tolerance and insulin sensitivity in leptin-deficient mice. After collection, MEFs were pretreated with the Nrf2 activator, dithiol-3-thione (D3T; 10 μM) for 12 h, and then treated with either hydrogen peroxide (0-2000 μM) or mercury (0-100 μM) for another 24 h. Viability was measured via MTT assay after 24 h of treatment. Implantation of gut-derived cells, engineered with a mifepristone-inducible leptin expression construct, has also been used to help improve metabolic effects in ob/ob mice. Graphs were plotted in GraphPad Prism 7.0e (GraphPad Software, La Jolla, CA), and data are expressed as mean ± SD. With the rise of genetically engineered animals, it is now common to confirm in vitro data acquired using immortalized cell lines with more physiologically relevant primary cells from these animals ex vivo. 2009 Dec 10;424(3):491-500. doi: 10.1042/BJ20091286. Nat Immunol 5:730–737, Kawai T, Takahashi K, Sato S et al (2005) IPS-1, an adaptor triggering RIG-I- and Mda5-vmediated type I interferon induction. Four male obR mice, at 3 to 4 months of age, were bred with WT females, resulting in litters within 1 month in all four cages. In addition, obR mice had a 26% lower food intake [Fig. the rationale for this is that my protein is significantly recurrently mutated in several cancers (from recent genomic papers). 7(b)].

Takahashi K., and Yamanaka S., (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Additionally, compared with adenoviral leptin production, daily treatment with recombinant leptin achieved a far greater decrease in BW over time (25), which showed a similar trend to what we observed in our obR mice 3 weeks after MEF implantation (Fig. (2008) Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF. Obese ob/ob mice are rescued with MEF implantation. However, their utility can extend well beyond their use exclusively in ESC culture.